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CD16+NK-92 and anti-CD123 antibody therapy for AML
NK-cell preparations. Another approach is to use a perma-
nent NK cell line, such as NK-92 which was derived from
a patient with an NK-cell lymphoma,10 and demonstrates
enhanced cytotoxicity over endogenously-derived NK
cells against a variety of human leukemia cell lines and pri-
mary leukemic blasts.11 However, this cell line lacks the Fc
gamma receptor IIIA (CD16), typically expressed by NK
cells and, therefore, cannot mediate antibody-dependent
cell-mediated cytotoxicity (ADCC). NK-92 has been test-
ed in three published phase I clinical trials, including one
clinical trial by our group for relapsed and refractory
hematologic cancers (lymphoma and multiple myeloma),
which all demonstrated minimal toxicity.12-14 However, to
prevent potential engraftment of NK-92 and generate a
NK malignancy, the cells are irradiated with 1000 cGy
which does not significantly decrease in vitro cytotoxici- ty.15-17
Natural killer cells typically express CD16 and are able to mediate ADCC against antibody-coated targets, enabling both adaptive and innate immune responses. Since the parental NK-92 cell line lacks CD16, and cannot mediate ADCC, a high-affinity allelic variant (valine at position 176 instead of phenylalanine) of the CD16A Fcγ receptor was transduced into the NK-92 cell line. These gene-modified CD16+NK-92 cells (NK-92.176V and NK- 92.176V.GFP) demonstrate ADCC in vitro18 but have not been tested in vivo.
Here, we show that NK-92 preferentially kills leukemic stem cells compared with bulk leukemia cells and can pro- long survival with or without prior radiation. Moreover, gene-modified NK-92 expressing the high affinity CD16 receptor (NK-92.176V.GFP) more effectively killed CD123+ targets in vitro and demonstrated an enhanced ability to target LSCs. Finally, irradiated CD16+NK-92 combined with the anti-CD123 antibody, 7G3, enhanced survival in a primary AML xenograft model compared with control arms.
Methods
Cell lines and primary samples
K562 was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in IMDM+10% FBS. OCI/AML2, OCI/AML3 and OCI/AML5 were generously provid- ed by Dr. Mark Minden and maintained in MEMalpha+ 10%FBS (OCI/AML2 and OCI/AML3) or MEMalpha+10% FBS and 10% 5637 bladder carcinoma conditioned medium (OCI/AML5). NK- 92 was originally kindly provided by Dr. Hans Klingemann, expanded and was maintained in X-VIVO 10 medium (Lonza) supplemented with 450 U/mL of IL-2 and 2.5% human AB serum (GM1). Four primary AML samples were obtained from the Princess Margaret Hospital Leukemia Tissue Bank, Toronto, Canada, according to an approved institutional protocol. NK-92 and NK-92.176V GFP (hereafter referred to as CD16+NK-92) was obtained from Conkwest under a Material Transfer Agreement (MTA) and maintained as described for NK-92. Frozen master cell banks for cell lines were established and new vials utilized to establish new cultures every six weeks. Mycoplasma testing by PCR was conducted periodically with all cultures testing negative.
Chromium release assay
We used a chromium release assay (CRA) as previously described by our group19 and detailed in the Online Supplementary Methods.
Flow cytometry and cell sorting
Immunophenotyping of bone marrow (BM) was performed using an FC500 or Facscalibur flow cytometer. FACS buffer was made with PBS+2mM EGTA+2% FBS. Primary AML and leukemic stem cell fractions were detected using the following antibodies (company; product #; clone): anti-CD34 PE (BD bio- sciences; 348057; 8G12), anti-CD34 FITC (BD Pharmingen; 555821; 581), anti-CD38 APC (ebiosciences; 17-0389-42; HIT2) and anti-CD123 PE (BD Pharmingen; 558714; 7G3), anti-CD45 APC (BD Pharmingen; 557513; TU116) and anti-class I HLA A, B, C (Biolegend; 311404; W6/32). NK-92 cells lines were assessed for CD16 expression using CD16 PE (Biolegend; 302008; 3G8). Leukemia cell lines were evaluated using anti-CD123 PerCy5.5 (BD Biosciences; 560904; 7G3). Cell sorting was performed using a FacsAria cell sorter as described in the Online Supplementary Methods.
Methylcellulose cytotoxicity assay
We used a methylcellulose cytotoxicity assay as previously described19 and included in the Online Supplementary Methods and in Supplementary Figure S1. Briefly, cell line or primary AML cells were incubated with and without NK-92 in a 4-hour assay prior to infusion into methylcellulose. Colonies were assessed at 2-4 weeks and percent colony inhibition calculated. NK-92 did not grow colonies under these conditions.
Animals
NOD/SCID gammanull (NSG) mice from The Jackson Laboratory were bred and maintained in the Ontario Cancer Institute animal facility according to protocols approved by the Animal Care Committee. Mice were fed irradiated food and Baytril containing water ad libitum during experimental periods. Prior to infusion with AML, NSG mice were irradiated with 325 or 225 cGy to facilitate engraftment. We developed a primary AML xenograft model utilizing a patient-derived AML sample (details in the Online Supplementary Methods and Supplementary Figure S2). Mice were sacrificed when humane end points were reached as per our animal use protocol (2791). Primary AML xenografted mice were treated with NK-92 (kindly provided by Hans Klingemann) or CD16+NK-92 (provided by Conkwest), with or without murine antibody therapy as follows: 7G3 (provided to RMR under an MTA with CSL Ltd., Parkville, Australia), BM4 isotype control (under MTA to RMR) and MG2a-53 isotype control (Biolegend; 401502; MG2a-53).
Statistical analysis
Survival analysis was carried out with Kaplan-Meier survival curves using the log rank rest (P<0.05) with Medcalc software. Comparison of cytotoxicity was made with the two-tailed Student t-test (P<0.05) to compare in vitro cytotoxicity and engraft- ment data using Medcalc software.
Results
NK-92 preferentially kills leukemic stem cells compared with bulk leukemia cells
We initially set out to determine the cytotoxicity of NK- 92 against primary AML cells using chromium release as a measure of bulk tumor cell kill. A panel of 4 primary AML blast samples treated with NK-92 yielded a dose-depen- dent response and moderate degrees of cytotoxicity against 4 samples at a 25:1 E:T ratio (% lysis): 080179 (42.3±3.6%), 080078 (29.8±3.6%), 08008 (43.9±1.47%), 0909 (42.6±0.1%) (Figure 1A). Primary AML cells were
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